Lack of interaction between ErbB2 andinsulin receptor substrate signaling inbreast cancerSusan M. Farabaugh1†, Bonita T. Chan2†, Xiaojiang Cui2, Robert K. Dearth2 and Adrian V. Lee1,2*
Abstract
Background: ErbB2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/Neu) is amplified in breast cancer and associated withpoor prognosis. Growing evidence suggests interplay between ErbB2 and insulin-like growth factor (IGF) signaling.For example, ErbB2 inhibitors can block IGF-induced signaling while, conversely, IGF1R inhibitors can inhibit ErbB2action. ErbB receptors can bind and phosphorylate insulin receptor substrates (IRS) and this may be critical for ErbB-mediated anti-estrogen resistance in breast cancer. Herein, we examined crosstalk between ErbB2 and IRSs usingcancer cell lines and transgenic mouse models.
Methods: MMTV-ErbB2 and MMTV-IRS2 transgenic mice were crossed to create hemizygous MMTV-ErbB2/MMTV-IRS2 bigenic mice. Signaling crosstalk between ErbB2 and IRSs was examined in vitro by knockdown oroverexpression followed by western blot analysis for downstream signaling intermediates and growth assays.
Results: A cross between MMTV-ErbB2 and MMTV-IRS2 mice demonstrated no enhancement of ErbB2 mediatedmammary tumorigenesis or metastasis by elevated IRS2. Substantiating this, overexpression or knockdown of IRS1or IRS2 in MMTV-ErbB2 mammary cancer cell lines had little effect upon ErbB2 signaling. Similar results wereobtained in human mammary epithelial cells (MCF10A) and breast cancer cell lines.
Conclusion: Despite previous evidence suggesting that ErbB receptors can bind and activate IRSs, our findingsindicate that ErbB2 does not cooperate with the IRS pathway in these models to promote mammary tumorigenesis.
Keywords: IRS, ErbB2, Breast cancer
BackgroundInsulin receptor substrate (IRS) proteins are cytoplasmicadapters which function as signaling intermediatesdownstream of cell surface receptors. Although IRS pro-teins are common intermediates of multiple growth andhormone receptors [1–6], they are most well known assignaling intermediates for the insulin receptor (InsR)and the insulin-like growth factor I receptor (IGF1R) [2,7–9]. IRS1 and IRS2 are expressed in normal andcancerous breast epithelium [6, 8, 10]. IRS1 and IRS2contain high homology and activate common signaling
pathways, such as PI3K/Akt and MAPK/ERK kinases[11], yet these two proteins have distinct functions [12].Generally, IRS1 is associated with tumor initiating pro-grams such as growth and survival while IRS2 is associatedmore closely with progression and metastasis [13–15]. AsIRS2 is the more definitive mediator of tumor progressionand metastasis, we focused our studies on IRS2.It is becoming increasingly evident that the IRS proteins
are regulated by epidermal growth factor receptor (EGFR)and Erb-B2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/Neu) [15–18]. For example, IRS2 levels are increased byEGFR and ErbB2 [15, 18]. Further, stimulation of cellswith EGF enhances IRS phosphorylation and downstreamPI3K signaling in the absence of IGF signaling [15, 19, 20].In tamoxifen-resistant breast cancer cells, EGFR andErbB3 recruit and phosphorylate IRS1 [16, 17].
* Correspondence: [email protected]†Equal contributors1Women’s Cancer Research Center, Department of Pharmacology andChemical Biology, University of Pittsburgh Cancer Institute, Magee Women’sResearch Institute, 204 Craft Avenue, Room A412, Pittsburgh, PA 15213, USA2Lester and Sue Smith Breast Center, Department of Molecular and CellularBiology, Baylor College of Medicine, Houston, TX 77030, USA
Given the emerging evidence for interaction betweenEGFR/ErbB2 and IGF/IRS signaling in breast cancer, we in-vestigated crosstalk between ErbB2 and IRSs. Herein, wedemonstrate that overexpression of IRS2 in MMTV-ErbB2transgenic mice did not alter mammary tumorigenesis ormetastasis. Consistent with this, overexpression or knock-down of IRS1 and IRS2 had little or no affect upon ErbB2action in both mouse and human mammary epithelial andbreast cancer cells. Taken together, our data indicates littleto no role for IRSs in ErbB2 action in breast cancer.
MethodsMaterialsAll chemicals were purchased from Sigma unless other-wise indicated. All tissue culture materials were purchasedfrom BD Falcon and Invitrogen unless otherwise stated.
Cell cultureThe BRI-JM04 mouse cell line was maintained inDMEM (with glutamine, glucose, sodium pyruvate) with10 % serum. The MCF-10A human mammary epithelialcell was maintained in DMEM/F-12 supplemented with5 % horse serum, EGF, chlorea toxin, hydrocortisone andinsulin according to ATCC standards.
Transient transfectionBRI-JMO4 cells were plated at 25–30 % density one daybefore transfection in antibiotic free complete medium.3.6x105 cells were plated per well of a 6 well plate. Foroverexpression experiments, the next day, cells weretransfected with pcDNA3.1 plasmids containing eitherHA-IRS1, HA-IRS2, or an empty vector control withLipofectamine 2000 (Thermo Fisher Scientific). For knock-down experiments, the next day, cells were transfected with50uM of siRNA against IRS1, IRS2, or both IRS1 and IRS2together using DharmaFECT 1 (Dharmacon). Transfectionswere performed following the instructions provided by themanufacturer.
Growth assayTwenty-four hours after siRNA transfection, BRI-JO4cells were washed and left to rest for four hours. 2,000cells per well were then seeded into 96-well plates andstarved overnight. After starvation, cells were treatedwith or without 10 % fetal bovine serum. Plates werewashed with PBS and frozen on days 1 and 4. When allof the plates were collected, cell growth was examinedby CyQuant (Thermo Fisher Scientific).
ImmunoblottingCells were lysed in RIPA lysis buffer 48 h after transfection.Protein concentration was determined using the BCA kit(Pierce). Twenty-five to 50 ug of protein was loaded ontoSDS-PAGE gels for analysis. Primary antibodies of HA (Cell
Signaling #3724S), pIGF-IR (Biosource #44804), IGF1R(Cell Siganling #9750), pY877-ERBB2 (Cell Signaling#2241), ERBB2 (Cell Signaling #2248), IRS1 (Upstate #06-248), IRS2 (Upstate #06-506), pAKT (Cell Signaling 9272),AKT (Cell Signaling 4060), pERK1/2 (Cell Signaling 4377),ERK1/2 (Cell Signaling 9102S) were diluted in 5 % BSAblocking buffer. Generation of ErbB2/IRS2 Bigenic Mice.All procedures were conducted in accordance with the
NIH Guide for the Care and Use of Laboratory Animalsand were approved by the IACUC at Baylor College ofMedicine. MMTV-ErbB2 mice were received fromJackson Laboratories [21]. MMTV-IRS2 mice have beenpreviously reported [5]. MMTV-ErbB2 mice were main-tained homozygous and MMTV-IRS were hemizygous.Mice were maintained on a 12-h light, 12-h dark schedulewith ad libitum access to laboratory chow and water. Togenerate bigenic mice, homozygous MMTV-ErbB2 malemice were bred with hemizygous MMTV-IRS2 femalemice to generate hemizygous MMTV-ErbB2 and hemizy-gous MMTV-ErbB2/MMTV-IRS2 bigenic mice. To studythe effect of parity, hemizygous MMTV-ErbB2 and hemi-zygous MMTV-ErbB2/MMTV-IRS2 bigenic female micewere bred with outbred CD1 male mice and allowed to gothrough a full pregnancy, lactation, and involution. Atweaning pups were removed and euthanized.
Analysis of median time to tumor formation (MTTF)Median times to tumor formation (MTTF) was deter-mined by weekly palpation. Tumor formation was re-corded when tumors were first palpable. When tumorsreached 1,000 mm3, tumors were harvested for molecu-lar analysis. Tumor volumes were measured withcalipers and volumes were calculated using the formulaVolume = (Length ×Width ×Width)/2.
HistologyFive um serial tumor sections were deparaffinized, grad-ually hydrated, and stained for hematoxylin and eosin(H&E). Sections were then examined and scored by apathologist to determine histological phenotypes as wellas stromal, inflammatory, and lactation properties.For the detection of lung metastases, 5 um sections
were cut at intervals of 100 um through one half of thelung, all sections were stained by H&E, and then exam-ined microscopically. Lungs were scored positive forlung metastases if they contained lesions of more than100 cells. All lesions were stained for HA (HA-IRS2) byIHC.
ResultsIRS2 overexpression does not affect ErbB2-mediatedtumorigenesis and metastasis in transgenic miceRecent evidence suggests that ErbB2 may utilize IRSs forsignaling. To determine potential cooperation between
Farabaugh et al. Cell Communication and Signaling (2016) 14:25 Page 2 of 7
these pathways, we analyzed the effect of elevated IRS2 onErbB2-mediated breast tumorigenesis. We comparedmammary tumorigenesis between MMTV-ErbB2 andMMTV-ErbB2/MMTV-IRS2 bigenic mice. Under parous(p) and nulliparous (np) conditions, median time to tumorformation (MTTF) was similar for both ErbB2 (p = 214and np = 244 days) and ErbB2/IRS2 bigenic mice (p = 211and np = 264 days) (Fig. 1a and b). Histological analysisrevealed that the bigenic ErbB2/IRS2 tumors closelymirrored the phenotype of ErbB2 tumors with 88 % solidadenocarcinoma in ErbB2 and 66 % solid adenocarcinomain ErbB2/IRS2 bigenic tumors (Fig. 1c and Table 1). Al-though some bigenic tumors (21 %) did show features ofMMTV-IRS2 mouse tumors such as squamous differenti-ation [5], most bigenic tumors recapitulated ErbB2 tumorphenotypes, suggesting that the ErbB2 pathway may be
the primary driver of tumorigenesis in these bigenic mice.We also noted no difference in lung metastasis in ErbB2and bigenic ErbB2/IRS2 mice with nodules observed at 53and 50 % for ErbB2 and bigenic mice, respectively (Fig. 1dand Table 1). Protein analysis of the tumors confirmedoverexpression of HA-tagged IRS2 in the bigenic ErbB2/IRS2 tumors (Fig. 1e). No difference in IRS1 levels werenoted. Interestingly, ErbB2 levels were decreased in bigenictumors.
Modulation of IRS1 and IRS2 levels in ErbB2-expressingmouse cells has little effect upon ErbB2 signaling and cellgrowthAs overexpression of IRS2 showed an unexpected lackof influence on ErbB2-driven tumorigenic phenotypes(Fig. 1), we further evaluated the interaction using in
Fig. 1 ErbB2 and ErbB2/IRS2 mice had similar time to tumor formation, tumor histology, and lung metastases. Kaplan-Meier plots of age-matchedMMTV-ErbB2/IRS2 bigenic (black line) and MMTV-ErbB2 transgenic (gray line) (a) parous and (b) nulliparous mice. Mean time to tumor formation (MTTF) isshown in days and was measured by weekly palpation. Formation was recorded when first palpable. c H&E of tumors from nulliparous mice representingthe histological phenotypes presented. d Immunoblotting of protein lysates from ErbB2 and ErbB2/IRS2 bigenic tumors for expression of IRS1, IRS2, HER2,and downstream signaling pathways. e Lungs of tumor-bearing mice were sectioned, stained with H&E, and analyzed for lung metastases. Numbersrepresent percentage of mice containing lung lesions to total number of mice analyzed
Farabaugh et al. Cell Communication and Signaling (2016) 14:25 Page 3 of 7
vitro culture models. As the functions and roles for IRS1and IRS2 are still unclear, we analyzed the effect of bothIRSs. To determine modulation of ErbB2 signaling uponoverexpression of IRS1 and IRS2 in vitro, HA-taggedIRS1 or HA-tagged IRS2 were transiently transfectedinto the MMTV-ErbB2 mouse cell line BRI-JM04.Knowlden et al. demonstrated that, in ER+ cells, heregu-lin (HRG) stimulates the recruitment of IRS1 to ErbB3[16, 17] and thus we examined the effect of HRG inBRI0-JM04 cells with or without IRS overexpression. Asshown in Fig. 2a, overexpression of IRS1 or IRS2 in BRI-JM04 cells in SFM (vehicle) conditions did not affectphosphorylation of IGF1R, Akt, or ERK1/2. HRG andIGF1 treatment increased phosphorylation of Akt andERK1/2 in BR10-JM04 cells, but overexpression of IRS1 orIRS2 (noted by HA expression) had little to no effect uponHRG or IGF1 induced Akt or ERK1/2 phosphorylation. If
anything, we noted a reproducible but very small decreasein HRG-induced Akt activity.To determine if IRS1 or IRS2 are required for ErbB2
signaling, we knocked-down each IRS individually andtogether in ErbB2 expressing BRI-JM04 cells and thenstimulated cells with HRG or EGF. siRNA knockdown ofIRS1 and IRS2 were both efficient and specific. Incontrol cells, HRG induced p-Akt, whereas EGF inducedp-ERK1/2. Knockdown of IRS1, IRS2 or the combinationcaused a slight decrease in HRG-induced p-Akt. Nomajor differences were observed with EGF treatmentswith or without IRS1/IRS2 knockdown. We next testedwhether the minor effect of IRS knockdown on p-Aktmay reduce cell growth; however, knockdown of IRS1 orIRS2 followed by a growth assay showed no significanteffect on cells grown in SFM or 10 % serum (Fig. 2c).
Elevated IRS1 and IRS2 levels in human breast cells doesnot affect EGFR or ErbB2 signalingAs the previous experiments were all performed usingmurine models, we explored the effect of IRS1 and IRS2on ErbB2 signaling in human immortalized non-transformed human mammary epithelial cells (MCF10A)which overexpress IRS1 or IRS2 and have previouslybeen reported to have enhanced IGF signaling [5].Stimulation of MCF10A cells with HRG or EGF resultedin phosphorylation of ErbB2, Akt and ERK1/2. Cellsoverexpressing IRS1 or IRS2 had increased basal phos-phorylation of Akt that was not affected by HRG or EGFtreatment. The basal phosphorylation of ERK1/2 wasalso elevated by IRS overexpression, but in this case theinduction (both fold induction and total phosphorylated
Table 1 Histological analysis of the ErbB2/IRS2 bigenic tumorsclosely mirror ErbB2 single transgenic tumors
ErbB2 ErbB2/IRS2
25 Tumors-18 mice 24 Tumors-17 mice
Histological Types
Solid Adenocarcinoma 88 % (22/25) 66 % (16/24)
Differentiated
Squamous Carcinoma 0 4 % (1/24)
Adenosquamous Carcinoma 8 % (2/25) 21 % (5/24)
Pillary 0 4 % (1/24)
Histological analysis of nulliparous and parous tumors were combined for atotal of 24 ErbB2/IRS2 bigenic tumors (18 mice) and 25 age-matched ErbB2transgenic tumors (17 mice). All tumor phenotypes were scored by a pathologistbased on H&E staining
Fig. 2 Modulation of IRS1 and IRS2 levels in ErbB2 expressing mouse cells alters ErbB2 signaling but not cell growth. a BRI-JM04 mouse cells werestarved overnight and then treated in the morning with either 60 ng/ul of heregulin (HRG) or 20 ng/ul of IGF1 for 15 mins and then harvested.Expression of ErbB2 signaling proteins was determined by immunoblotting. Blots are representative of 3 experiments. b BRI-JM04 cells were transfectedwith siRNA against IRS1, IRS2, or both IRS1 and IRS2. At 48 h post-transfection, cells were stimulated with 20 ng/ul heregulin (H) or 60 ng/ul EGF (E) for15 min. Cells were then harvested and analyzed for ErbB2 signaling by immunoblotting. c After IRS1 or IRS2 knockdown, BRI-JMO4 cells were platedand maintained with or without serum for 4 days. Cell growth was determined by CyQuant. Graph is representative of 3 experiments. One-way Anovawas applied. N.S. = not significant
Farabaugh et al. Cell Communication and Signaling (2016) 14:25 Page 4 of 7
levels) by EGF and HRG was lower than in MCF10Acells (Fig. 3).
DiscussionThe IGF and ErbB pathways are involved in tumor initi-ation, tumor progression and resistance to therapy [22–25].These pathways overlap in their signal transduction path-ways, sharing PI3K/Akt and MEK/ERK signaling, and sev-eral studies suggest crosstalk between the pathways may beat least partially responsible for resistance to both ErbB2-and IGF1R-targetered therapies [25–31]. Several studieshave previously demonstrated that EGFR/ErbB recruits andactivates IRSs [16–18], and in some cases suggests a processfor therapeutic resistance to drugs such as tamoxifen. In thisreport we examined the role of IRSs in ErbB2 action in vitroand in vivo. Contrary to expected, we demonstrate that in-creased IRS1 or IRS2 expression does not enhance the abil-ity of ErbB2 to further stimulate downstream signalingpathways and enhance tumorigenic phenotypes.In this study, we hypothesized that increasing both
ErbB2 and IRSs would result in enhanced downstreamsignaling (through Akt and ERK1/2). However, in our invitro cell line data, we observed that increased ordecreased IRS levels had little to no effect upon ErbB2signaling. It should be noted that our studies were per-formed in the presence of overexpressed ErbB2 whichwas developed to mimic the amplification seen in hu-man breast cancer. Other studies have noted ErbB2-IRSinteraction and cooperation in antiestrogen resistancewhere the ErbB2 is simply elevated and not amplified. Itis possible that in our model systems, ErbB2 is overex-pressed at a level where it no longer requires IRS expres-sion beyond what is endogenously expressed or the levelat which ErbB2 is able to induce. This does not rule out
a role for the ErbB2-IRS cooperation in antiestrogen re-sistance, but suggests that ErbB2 amplified human breastcancers may have sufficient ErbB2-induced regulationand signaling. It is also possible that the overexpressionof ErbB2 has disrupted normal signaling networks andinteractions. Recent studies have shown an intricate net-work of feedback mechanisms with PI3K/S6K regulatingIGF1R/IRSs and MEK negatively feeding back to theErbB receptors [32, 33]. There is thus a fine balance ofreceptor signaling components with interferencepossible when one is overexpressed. Limited amounts ofsubstrates can also cause squelching. For example, re-cruitment of IRSs to alternate receptors such as theEGFR family limits the amount of IRS available for asso-ciation with IGF1R which in turn limits downstreamIGF1R signaling [16]. Inhibition of the EGFR pathwaycan then direct signaling back through the IGF1R-IRSassociation [16].In vivo, overexpression of IRS2 did not alter ErbB2 me-
diated tumorigenesis. Interestingly, ErbB2 tumors showedsimilar histologic phenotypes (solid adenocarcinomas)compared to bigenic ErbB2/IRS2 tumors, suggesting thateven in the presence of elevated IRS2, the ErbB2 signalingpathway remains the major driver of tumorigenesis. Wedid identify a small amount of squamous metaplasia inbigenic tumors, something that is rarely seen in ErbB2tumors, but widely observed in IRS2 tumors; however, thiseffect was minimal and rarely seen.
ConclusionIn conclusion, we find little evidence that increased IRSexpression enhances ErbB2 mediated signaling andtumorigenesis. This does not corroborate or conflictwith other studies as our models may have sufficient IRS
Fig. 3 Modulation of IRS1 and IRS2 expression in human MCF10A breast cells alters ErbB2 signaling. MCF10A cells stably overexpressing HA-IRS1or HA-IRS2 were serum starved for 24 h and then treated with 20 ng/ul heregulin (H) or 60 ng/ul EGF (E) for 15 mins and harvested. Immunoblottingwas performed to determine ErbB2 signaling
Farabaugh et al. Cell Communication and Signaling (2016) 14:25 Page 5 of 7
expression to support ErbB2 signaling. We show thisphenotype in both human and murine models, in vitroand in vivo. A better understanding of this complex sys-tems network will be critical to optimize response toanti-growth factor receptor and signaling intermediate(e.g. PI3K, mTOR) inhibitors in breast cancer.
AcknowledgementsWe would like to thank Dr. Shivendra Singh for the BRI-JM04 cells and Ms.Ora Britton for animal husbandry.
FundingResearch reported in this publication was supported in part by the National CancerInstitute of the National Institutes of Health under award numbers R01CA94118 andP30047904. The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes of Health. AVL is arecipient of a Susan G. Komen Scholar award (SAC150021). SMF is a recipient of aDepartment of Defense Breast Cancer Research Postdoctoral Fellowship award(W81XWH-14-1-0063). The authors acknowledge support from the University ofPittsburgh Cancer Institute (UPCI) and UPMC.
Availability of data and materialsNot applicable.
Authors’ contributionsXC developed the transgenic mice and initiated the study. RD carried out mousebreeding, harvesting, and analysis of tumors. BC performed in vitro experimentsand drafted the original manuscript. SF performed in vitro experiments andre-drafted and revised the manuscript. AL conceived the study, participated in itsdesign and coordination, and revised the manuscript. All authors read and approvethe final manuscript.
Competing interestsThe authors declare that the research was conducted in the absence of anycommercial, financial, or non-financial interests in the subject matter ormaterials discussed in this manuscript that could be construed as a potentialconflict of interest.
Consent for publicationNot applicable.
Ethics approval and consent to participateAll animal procedures were conducted in accordance with the NIH Guide forthe Care and Use of Laboratory Animals and were approved by the IACUC atBaylor College of Medicine. MMTV-ErbB2 mice were received from JacksonLaboratories [21].
Received: 5 February 2016 Accepted: 13 October 2016
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